Alkylphosphonoserines and pharmaceutical compositions useful as cytostatic agents

ABSTRACT

Compounds of formula I ##STR1## in which R denotes a straight-chain or branched, saturated or unsaturated aliphatic hydrocarbon radical having 6-30 C atoms wich may optionally be substituted by halogen or the radicals OR 1 , SR 1  or NR 1  R 2 , where R 1  and R 2  in each case denote a hydrogen atom or an alkyl or acyl radical having 1-6 C atoms which have excellent virustatic and cytostatic action.

DESCRIPTION

The invention relates to novel alkylphosphonoserines, processes fortheir preparation and their use.

A process for the preparation of alkylphosphocholines is described in R.Hirt, R. Berchthold Pharmaceutica Acta Helvetica, 33 (1958), 349-356 Thecytostatic activity and the toxicity of hexadecylphophocholine isdescribed in H. R. Scherf et al., Lipids 22, (1987), 927-929, and in C.Muschol et al. Lipids 22, (1987) 930-934.

It has now been possible to find novel alkylphosphonoserines which havean excellent cytostatic and virustatic action.

The invention relates to compounds of the formula I ##STR2## in which Rdenotes a straight-chain or branched, saturated or unsaturated aliphatichydrocarbon radical having 6-30 C atoms which may optionally besubstituted by halogen or the radicals OR₁, SR₁ or NR₁ R₂, where R₁ andR₂ in each case denote a hydrogen atom or an alkyl or acyl radicalhaving 1-6 C atoms.

In the formula I, R denotes a straight-chain or branched, saturated orunsaturated aliphatic hydrocarbon radical having 6-30 C atoms,preferably having 10-25 C atoms. Examples of such radicals are decyl,dodecyl, tetradecyl, hexadecyl, octadecyl, arachyl and17-methyl-n-octadecyl radicals or the cis-9-n-octadecenyl radical.

These radicals may also be substituted by halogen, for example by Cl,Br, F or by the radicals OR₁, SR₁ or NR₁ R₂. R₁ and R₂ in this caseindependently of one another denote hydrogen or an alkyl or acyl radicalhaving 1-6 C atoms, for example a methyl, ethyl, n-propyl, i-propyl,n-butyl, i-butyl, pentyl or hexyl radical or, for example, a formyl,acetyl, propionyl, butyryl or i-butyryl radical.

The alkylphosphonoserines have certain structural relationships tophysiologically active naturally occurring phospholipids, for examplephosphatidylserines. However, they differ from these in that theycontain no glycerol component. In comparison to the naturally occurringphosphatidylserines, the alkylphosphonoserines according to theinvention have a higher biostability. They are potential cytostatics andvirustatics.

The novel alkylphosphonoserines can be prepared by a process in which

a) a compound of the formula II ##STR3## in which R has theabovementioned meaning and A and B denote a hydroxyl group or, ifdesired, the salts of these compounds are reacted with a serinederivative of the formula III ##STR4## in which X denotes benzyl,t-butyl, phthalimidomethyl, isopropyl or benzhydryl, Y denotesN-benzoyloxycarbonyl, N-t-butoxycarbonyl or N-phthaloyl, if appropriatein the presence of a condensing agent, to give compounds of the formulaIV ##STR5## the protecting groups are removed and, if desired, theproduct is converted into a salt or

b) a compound of the formula V ##STR6## in which R has theabovementioned meaning and Z denotes an alkyl radical having 1-6 Catoms, which may optionally be substituted by Cl, Br or F or denotes theradicals ##STR7## in which C and D independently of one another denote Hor methyl and E denotes methyl and n denotes 2-6, is reacted withL-serine in the presence of phospholipase D.

The compounds of the formula II used as starting compounds can beprepared in a manner known per se by first synthesizing the dimethylalkylphosphonates from the corresponding alkyl bromide R-Br andtrimethyl phosphite, converting these into the trimethylsilyl esters byreaction with a mixture of trimethylsilyl chloride and sodium iodide andhydrolyzing these by the action of water.

The reaction of the compounds of the formula II is in general carriedout by converting the compounds into the salt of a base, for examplepyridine, and then reacting this salt with the serine derivative. Thereaction is preferably carried out in the presence of a condensingagent, for example in the presence of 2,4,6-triisopropylbenzenesulphonylchloride under anhydrous conditions and preferably in the presence of aninert organic solvent. A suitable solvent is, for example, pyridine.

The reaction components are customarily employed in equimolar amounts,but is also possible to employ up to a four molar excess of serinederivative.

The reaction is carried out at temperatures of about 5°-60° C.,preferably at room temperature. After completion of the reaction, theacid chloride still present is destroyed in a customary manner, forexample by adding water to the reaction solution, and the product isisolated in a customary manner, for example by extraction with diethylether. If desired, the compounds of the formula IV thus obtained can befurther purified before the removal of the protecting groups, forexample by column chromatography on silica gel. However, it is alsopossible to convert them without further purification into the compoundsof the formula I.

For this purpose, the protecting groups are removed in a customarymanner, for example by catalytic hydrogenolysis, hydrazinolysis ortreatment with HCl or formic acid, preferably in an inert solvent. Thecompounds of the formula I can then be isolated in a known manner and,if desired, further purified, for example by chromatography.

The alkylphosphonoserines according to the invention can also beprepared by an enzymatic route. In this connection, compounds of theformula V are reacted with L-serine in the presence of phospholipase D.For this purpose, a solution or suspension of the compounds of theformula V is reacted with an excess of L-serine in an aqueous buffersystem, for example in an acetate or tris buffer solution, at pH valuesof about 4-8.5, preferably 5-6, particularly preferably 5.6, in thepresence of about 0.01--0.1 M CaCl₂ and in the presence of phospholipaseD with the addition of diethyl ether or a mixture of diethyl ether witha further organic solvent, for example chloroform.

The reaction is carried out at temperatures of about 5°-60° C.,preferably 35°-45° C. The reaction mixture is intensively stirred orshaken during the reaction. The duration of the reaction is about 0.5-48hours. After reaction is complete, the enzyme phospholipase D isdeactivated, for example by addition of a 0.1M EDTA solution and thecompounds of the formula I are isolated in a customary manner.

The novel alkylphosphonoserines have a strong cytostatic and virustaticaction. In order to determine the cytostatic action, theantiproliferative action was investigated in Ehrlich ascites tumorcells. It can be seen here that the compounds according to the inventionhave excellent cytostatic action. They can therefore be employed ascytostatics or virustatics, alone or in a mixture with other activesubstances, in the form of customary pharmaceutical preparations.

The compounds of the formula I are intended for use in humans and can beadministered in a customary manner, for example orally or parenterally.They are preferably administered orally, the daily dose being about 0.05to 20 mg/kg of body weight, preferably 0.05-5 mg/kg of body weight.However, depending on the general conditions and the age of the patient,the corresponding compound of the formula I, the nature of the diseaseand the manner of formulation, the treating physician can also prescribelower or higher doses outside this range.

The compounds of the formula I can be administered alone or incombination with other pharmaceutically active substances, the contentof the compounds of the formula I being between 0.1 and 99%. In general,the pharmaceutically active compounds are present in a mixture withsuitable inert auxiliaries and/or excipients or diluents, for examplepharmaceutically acceptable solvents, gelatin, gum arabic, lactose,starch, magnesium stearate, talc, vegetable oils, polyalkylene glycol,petroleum jelly or the like. The pharmaceutical preparations can bepresent in solid form, for example as tablets, coated tablets,suppositories, capsules and the like, in semi-solid form, for example asointments or in liquid form, for example as suspensions or emulsions. Ifdesired, they are sterilized and contain auxiliaries such aspreservatives, stabilizers and emulsifiers, salt for changing theosmotic pressure and the like. Pharmaceutical preparations can inparticular contain the compounds according to the invention incombination with other therapeutically useful substances. The compoundsaccording to the invention can be formulated with these, for exampletogether with the abovementioned auxiliaries and/or excipients ordiluents, to give combination preparations.

EXAMPLE 1 n-Hexadecylphosphono-L-serine n-Hexadecylphosphonic acid

A mixture of n-hexadecyl bromide (4.89 g, 16 mmol) and freshly distilledtrimethyl phosphite (2.48 g, 20 mmol) is stirred at 116° C. for 24hours. The excess trimethyl phosphite is then removed by distillation invacuo and the residue is stored over phosphorus pentoxide in vacuo for24 hours. The corresponding dimethyl n-hexadecylphosphonate is isolatedfrom the residue by extracting twice with 10 ml of methanol each time.The two methanol extracts are combined and concentrated in vacuo. Theoily colorless residue thus obtained, which still contains n-hexadecylbromide in addition to the methyl n-hexadecylphosphonate, is furtherreacted with trimethylsilyl chloride (1.02 g, 9.4 mmol) and sodiumiodide (1.4 g, 9.4 mmol) in 4.7 ml of anhydrous acetonitrile. Thereaction is carried out with stirring at room temperature and lasts 15minutes.

The sodium chloride formed in the reaction is filtered off. The filtrateis concentrated in vacuo and 4 ml of water are added to the resultingresidue, n-hexadecylphosphonic acid being formed by hydrolysis. In orderto remove the Me₃ SiOSiMe₃ simultaneously formed, the mixture issubsequently distilled in vacuo several times with ethanol/water (1:1,v/v). After treatment of the residue with 7 ml of dry acetone, 346 mg ofn-hexadexylphosphonic acid II are obtained. TLC (silica gel 60, Merckprepared plates, chloroform/methanol/water 65:25:4, v/v/v): Rf=0.15.

Elemental analysis calculated for C₁₆ H₃₅ PO₃ (306.42): C 62.71, H11.51; found C 62.81, H 11.89 (%).

n-Hexadecylphosphono-N-t-butoxycarbonyl-L-serine benzhydryl ester

A solution of 92 mg (0.3 mmol) of n-hexadecylphosphonic acid in 1 ml ofpyridine is concentrated in vacuo and the pyridinium salt thus obtainedis subsequently dried over phosphorus pentoxide in vacuo. It is thendissolved in 5 ml of anhydrous pyridine. 356 mg (0.96 mmol) of protectedserine (X=CH(C₆ H₅)₂, Y=CO--O--C(CH₃)₃), dried over phosphorus pentoxidein vacuo, are dissolved in 4 ml of anhydrous pyridine. The two solutionsare mixed and 581 mg (1.92 mmol) of triisopropylbenzenesulfonyl chlorideare added to the mixture. The reaction mixture is stirred underanhydrous conditions at room temperature for 24 hours. The mixture isthen concentrated in vacuo and subsequently distilled several times withtoluene, and the residue is dried over phosphorus pentoxide in vacuo.The residue is then extracted with 15 ml of diethyl ether andsubsequently washed three times with 4 ml of diethyl ether. The ethersolutions are combined and concentrated in vacuo. 118 mg of then-hexadecylphosphonoserine derivative IV are obtained byrecrystallization from acetonitrile of the residue thus obtained. Afurther purification is carried out by column chromatography on 8 g ofsilica gel 60 (particle size 0.04-0.063 mm, 230-400 mesh, Merck).

The chloroform used for the chromatography and the solvent mixtures forthe elution contain 0.5% aqueous 25% strength ammonia. Elution iscarried out using 50 ml of chloroform, 50 ml of chloroform/methanol98:2, v/v, 200 ml of chloroform/methanol 95:5, v/v and 200 ml ofchloroform/methanol 90:10, v/v. The fraction size is 15 ml. Fractions15-20 contain 72 mg of purehexadecylphosphono-N-t-butoxycarbonyl-L-serine benzhydryl ester. TLC(silica gel 60, Merck prepared plates, chloroform/methanol 80:20, v/v):Rf=0.2.

Elemental analysis calculated for C₃₇ H₆₁ N₂ PO₇ (NH₄ salt, 676.87):

C 65.65, H 9.08, N 4.14; found C 65.44, H 9.15, N 3.73 (%).

n-Hexadecylphosphono-L-serine

69 mg (0.1 mmol) of the compound described are dried in vacuo overphosphorus pentoxide and subsequently dissolved in 13 ml of anhydrouschloroform. Dry nitrogen is passed through this solution for 10 minutesand hydrogen chloride gas is subsequently passed through the solutionunder anhydrous conditions at 0° C. for 20 minutes. The reaction vesselis then firmly closed and further stirred at 0° C. for 1 hour. In orderto remove the hydrogen chloride gas, nitrogen is subsequently passedthrough the reaction mixture for 1 hour at room temperature. Thereaction mixture is then concentrated in vacuo and a mixture of 10 ml ofchloroform/methanol 2:1, v/v, 1 ml of water and 0.02 ml of 25% strengthaqueous ammonia is then added to the residue, the mixture is shaken and,after phase separation, the organic phase obtained is separated off. Theaqueous phase is extracted three times using 5 ml ofchloroform/methanol/water 2:1:0.18, v/v/v, each time, and the organicsolutions are combined and concentrated in vacuo. In order to removeresidual water, the residue is subsequently distilled several times with1 ml of benzene/ethanol 2:3, v/v. The product obtained,hexadecylphosphono-L-serine I, is dried over phosphorus pentoxide invacuo and then washed with acetone.

Yield: 43 mg (95%).

In order to remove traces of ammonium chloride, the substance ischromatographed on 2.2 g of silica gel 60 (particle size 0.04-0.063 mm,230-400 mesh, Merck). The column is equilibrated usingchloroform/methanol/aqueous 25% strength ammonia 80:20:0.5, v/v/v.Elution is carried out using 20 ml of chloroform/methanol/aqueous 25%strength ammonia 80:20:0.5, v/v/v, 20 ml of chloroform/methanol/aqueous25% strength ammonia 75:25:0.5, v/v/v, 20 ml ofchloroform/methanol/aqueous 25% strength ammonia 70:30:0.5, v/v/v, 20 mlof chloroform/methanol/aqueous 25% strength ammonia 65:35:0.5, v/v/v and200 ml of chloroform/methanol/aqueous 25% strength ammonia 60:40:0.5,v/v/v. The compound is obtained from fractions 7-19 by concentration.(Fraction size 20 ml).

TLC (silica gel 60, Merck prepared plates,chloroform/methanol/acetone/acetic acid/water 10:2:4:2:1, v/v/v/v/v):Rf=0.1.

Visualization is carried out using molybdate reagent and using ninhydrinreagent.

Elemental analysis calculated for C₁₉ H₄₅ N₂ PO₆ (NH₄ salt, monohydrate,428.55):

C 53.24, H 10.58, N 6.54; found C 53.80, H 10.55, N 5.73 (%).

FAB mass spectrometry:

Anion spectrum: m/e 414, (M--2H+Na)⁻ ; m/e 392, (M--H)⁻ ; m/e 305(M--CH₂ CH(NH₂)COOH)⁻ ;

Cation spectrum: m/e 416, (M+Na)⁺ ; m/e 438, (M+2Na)⁺.

EXAMPLE 2 n-Hexadecylphosphono-L-serine

0.8 g of L-serine (7.6 mmol) is dissolved in 1.52 ml of 0.1M acetatebuffer (pH 5.6), which is 0.09M in CaCl₂, at 45° C. 31 mg (0.075 mmol)of n-hexadecylphosphonocholine, 1.6 ml of diethyl ether/chloroform (9:1,v/v, ethanol-free) and 200 mg of a phospholipase D enzyme preparationwhich has been prepared from white cabbage and has an activity of 0.9U/ml (1 unit (U) converts 1 μmol of substrate per minute at 27° C.) inthe reaction mixture, are added to this solution. The mixture is stirredat 45° C. for 2.2 hours. After cooling to room temperature, 3.31 ml of0.1M EDTA solution are added and the organic solvents are removed bypassing in nitrogen. The aqueous phase which remains is stirred with 4.3times its volume of chloroform/methanol (5:8, v/v) for 30 minutes andthe undissolved material (L-serine) is filtered off with suction. 1volume of water and 3.7 volumes of chloroform are added to the filtrate,the mixture is shaken for 10 minutes, the organic phase is separated offand concentrated in vacuo, and the residue obtained is separated bycolumn chromatography on carboxymethyl cellulose (CM 52 Whatman, Na⁺form) Elution is carried out successively with 75 ml of chloroform, 300ml each of chloroform/methanol 9:1, 8:2 and 7:3, then 1,750 ml ofchloroform/methanol 1:1 (in each case v/v). The fraction size is 50 ml.5 mg of pure n-hexadecylphosphono-L-serine (I, R=C₁₆ H₃₃) are obtainedfrom fractions 20-39.

TLC (silica gel 60, Merck prepared plates, CHCl₃ /CH₃ OH/acetone/aceticacid/H₂ O 50:10:20:10:5, v/v/v/v/v: Rf=0.1.

Further compounds were prepared in an analogous manner.

The following compounds were prepared analogously to Example 1.

EXAMPLE 3 n-Decylphosphono-L-serine

From n-decylphosphonic acid and protected serine (formula III, X=CH(C₆H₅)₂, Y=CO-OC(CH₃)₃. TLC (silica gel 60, Merck prepared plates,chloroform/methanol/acetone/acetic acid/water 10:2:4:2:1; v/v/v/v/v):Rf=0.1.

EXAMPLE 4 n-Dodecylphosphono-L-serine

From n-docecylphosphonic acid and protected serine (formula III, X=CH(C₆H₅)₂, Y=CO-OC(CH₃)₃. TLC (silica gel 60, Merck prepared plates,chloroform/methanol/acetone/acetic acid/water 10:2:4:2:1; v/v/v/v/v):Rf=0.1.

EXAMPLE 5 n-Tetradecylphosphono-L-serine

From n-tetradecylphosphonic acid and protected serine (formula III,X=CH(C₆ H₅)₂, Y=CO-OC(CH₃)₃. TLC (silica gel 60, Merck prepared plates,chloroform/methanol/acetone/acetic acid/water 10:2:4:2:1; v/v/v/v/v):Rf=0.1.

EXAMPLE 6 n-Octadecylphosphono-L-serine

From n-Octadecylphosphonic acid and protected serine (formula III,X=CH(C₆ H₅)₂, Y=CO--OC(CH₃)₃ TLC (silica gel 60, Merck prepared plates,chloroform/methanol/acetone/acetic acid/water 10:2:4:2:1; v/v/v/v/v):Rf=0.1.

EXAMPLE 7 Arachylphosphono-L-serine

From arachylphosphonic acid and protected serine (formula III, X=CH(C₆H₅)₂, Y=CO-OC(CH₃)₃. TLC (silica gel 60, Merck prepared plates,chloroform/methanol/acetone/acetic acid/water 10:2:4:2:1; v/v/v/v/v):Rf=0.1.

EXAMPLE A Concentration-dependent Inhibition of Growth of EhrlichAscites Tumor Cells

    ______________________________________                                        Concentration (M)                                                                         1 × 10.sup.-4                                                                    3 × 10.sup.-5                                                                    1 × 10.sup.-5                                                                  3 × 10.sup.-6                      Inhibition (%)                                                                n-Hexadecyl 89       45       13     9                                        phosphono-L-serine                                                            ______________________________________                                    

We claim:
 1. Compounds of the formula I ##STR8## in which R denotes astraight-chain or branched, saturated or unsaturated aliphatichydrocarbon radical having 6--30 C atoms which may optionally besubstituted by halogen or the radicals, OR₁, SR₁ or NR₁ R₂, where R₁ andR₂ in each case denote a hydrogen atom or an alkyl or acyl radicalhaving 1-6 C atoms.
 2. Compounds of the formula I ##STR9## according toclaim 1, in which R denotes a straight-chain or branched, saturated orunsaturated aliphatic hydrocarbon radical having 10-25 C atoms which mayoptionally be substituted by halogen or the radicals OR₁, SR₁ or NR₁ R₂,where R₁ and R₂ in each case denote a hydrogen atom or an alkyl or acylradical having 1-6 C atoms.
 3. n-Hexadecylphosphono-L-serine [sic].
 4. Apharmaceutical composition, containing compounds of the formula I##STR10## in which R denotes a straight-chain or branched, saturated orunsaturated aliphatic hydrocarbon radical having 6--30 C atoms which mayoptionally be substituted by halogen or the radicals OR₁, SR₁ or NR₁ R₂,where R₁ and R₂ in each case denote a hydrogen atom or an alkyl or acylradical having 1-6 C atoms, in combination pharmaceutical auxiliaries,excipients and/or diluents.